Recent studies have shown that Ca ions activation of cardiac myofibrils can be modulated by actin-myosin interactions (linkage-dependent modulation), by nucleotides, Mg ions and ionic strength (simple chemical modulation) and by phosphorylation of myofibrillar proteins (complex chemical modulation). The general objective of experiments proposed here is to make a detailed study of the relative role of such modulation as regulatory device in normal hearts and as a locus of lesions in anoxic or ischemic hearts. Specifically, I propose: (1) To investigate effects of alterations of numbers of connected cross-bridges on Ca ions binding, Mg ions binding, and energy transduction of cardiac myofibrils, (2) To investigate the nature of the interaction between Mg ions and myofibril which could account for our observations of positive cooperative interactions induced by this cation and other cations, (3) To investigate the effects of conditions (lactate, ADP and pH) which change during cardiac ischemia and anoxia on calcium binding and energy transduction of cardiac myofibrils, (4) To study the dynamics of troponon and myosin phosphorylation during positive and negative inotropic interventions in isolated and in situ beating hearts, and (5) To test whether phosphorylation of these proteins affects calcium activation, chemical modulation and linkage dependent modulation. Methods used in these studies are, for the most part, those which I have used in the past to measure calcium activation and energy transduction of heart myofibrils, but with emphasis on modulation. Preparation of proteins from single rat hearts will involve recently developed micro-procedures for preparation of troponin I and light chains or fragments of myosin by affinity chromatography. Working rat heart preparations will be used to determine whether phosphoryl group transfer reactions involving myofibrillar proteins are a link between myofibrils, events at the surface membrane, metabolic events and events associated with anoxic and ischemic hearts.